Multiple sexed embryo production system for bovine mammals

ABSTRACT

Improved insemination systems particularly adapted to use for sex-selected sperm sorting include systems which achieve superovulation and then multiple embryo production with sexed embryos. These systems combine with other techniques, including techniques for enhanced sheath fluid and other strategies which minimize stress on the sperm cells, and, potentially, a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems and techniques for the process are described so that commercial application of sperms samples as well as the resulting animals may be achieved.

This application is a continuation of U.S. application Ser. No.09/448,643, filed Nov. 24, 1999, now issued as U.S. Pat. No. 6,372,422which was a continuation of U.S. application Ser. No. 09/015,454, filedJan. 29, 1998, now issued as U.S. Pat. No. 6,071,689 which was acontinuation-in-part of U.S. application Ser. No. 09/001,394, filed Dec.31, 1997, now issued as U.S. Pat. No. 6,149,867, each herebyincorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of sex selection inmammalian offspring. It is especially relevant to the aspect of low doseartificial insemination for creating the desired sex of offspring.Particularly, the invention relates to systems for sorting sperm viaflow cytometry for sex-specific and low dose efforts at artificialinsemination or the like.

For ages it has been desired to select the sex of specific offspring.Beyond obvious psychological aspects, the actual sex selection ofmammalian offspring has significant economic s consequences when oneconsiders its application to food producing animals such as cattle aswell as celebrated trophy animals such as horses and the like. Thisgreat desire has resulted in a significant variety of efforts to achievesex-selected offspring. Probably the effort which has appeared mostlikely to achieve the desired results has been efforts at sorting andselecting between X and Y sperm prior to insemination.

One of the challenges that effort at sorting X and Y sperm has faced isthe large numbers of sperm involved. In natural insemination sperm areproduced in some species by the billions; in artificial inseminationless, but still significantly large numbers of sperm are used. Forinstance, artificial insemination techniques commonly use ten million tofive hundred million sperm (depending on species). Thus a significantnumber of sperm are necessary even in an artificial inseminationenvironment.

Many methods have been attempted to achieve the separation of X- andY-chromosome bearing sperm. These methods have ranged from magnetictechniques such as appears disclosed in U.S. Pat. No. 4,276,139 tocolumnar techniques as appears disclosed in U.S. Pat. No. 5,514,537 togravimetric techniques as discussed in U.S. Pat. Nos. 3,894,529, ReissuePat. No. 32350, U.S. Pat. Nos. 4,092,229, 4,067,965, and 4,155,831.Electrical properties have also been attempted as shown in U.S. Pat. No.4,083,957 as well as a combination of electrical and gravimetricproperties as discussed in U.S. Pat. No. 4,225,405, 4,698,142, and4,749,458. Motility efforts have also been attempted as shown in U.S.Pat. Nos. 4,009,260 and 4,339,434. Chemical techniques such as thoseshown in U.S. Pat. Nos. 4,511,661 and 4,999,283 (involving monoclonalantibodies) and U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and5,660,997 (involving membrane proteins), and U.S. Pat. Nos. 3,687,803,4,191,749, 4,448,767, and 4,680,258 (involving antibodies) as well asthe addition of serum components as shown in U.S. Pat. No. 4,085,205.While each of these techniques has been presented as if to be highlyefficient, in fact at present none of those techniques yield the desiredlevel of sex preselection.

At present, the only quantitative technique used to achieve theseparation of X- and Y-chromosome bearing sperm has been that involvingindividual discrimination and separation of the sperm through thetechniques of flow cytometry. This technique appeared possible as aresult of advances and discoveries involving the differential dyeabsorption of X- and Y- chromosome bearing sperm. This was discussedearly in U.S. Pat. No. 4,362,246 and significantly expanded upon throughthe techniques disclosed by Lawrence Johnson in U.S. Pat. No. 5,135,759.The Johnson technique of utilizing flow cytometry to separate X- and Y-chromosome bearing sperm has been so significant an advancement that ithas for the first time made the commercial separation of such spermfeasible. While still experimental, separation has been significantlyenhanced through the utilization of high speed flow cytometers such asthe MoFlo® flow cytometer produced by Cytomation, Inc. and discussed ina variety of other patents including U.S. Pat. Nos. 5,150,313,5,602,039, 5,602,349, and 5,643,796 as well as international PCT patentpublication WO 96/12171. While the utilization of Cytomation's MoFlo®cytometers has permitted great increases in speed, and while these speedincreases are particularly relevant given the high number of sperm oftenused, certain problems have still remained. In spite of the almostten-fold advances in speed possible by the MoFlo® flow cytometer,shorter and shorter sorting times have been desired for several reasons.First, it has been discovered that as a practical matter, the sperm aretime-critical cells. They loose their effectiveness the longer theyremain unused. Second, the collection, sorting, and insemination timingshas made speed an item of high commercial importance. Thus, the timecritical nature of the sperm cells and the process has made speed anessential element in achieving high efficacy and success rates.

Other problems also exist ranging from the practical to the theoretical.On the practical side, it has been desired to achieve sex-sorted spermsamples using inexpensive disposable components and substances. Also onthe expense side, it has been desired to be able to achieve sorting (aswell as collection and insemination) in as efficient a labor event aspossible. Thus, for commercial production and success in this field,improvements which might only represent an increase in efficiency maystill be significant. Related to the practical aspect of expense, is thepractical aspect of the delicateness and sensitivity of the entireprocess. In this regard, it has been desired to simplify the process andmake it as procedurally robust as possible so that operator error orskill can play an ever decreasing role.

In addition to the delicateness of the process, it has always been knownthat the sperm themselves are extremely delicate cells. While thisfactor at first glance seems like it might be considered easilyunderstood, in fact, the full extent of the cells' sensitivities havenot yet been fully explored. In the context of flow cytometry ingeneral, most sorted cells or particles have often been spherical orotherwise physically able to withstand a variety of abuses. This is notthe case for sperm cells. In fact, as the present invention discloses,the processing through normal flow cytometer techniques may, in fact, beunacceptable for cytometric sorting of sperm cells in certainapplications. The sensitivities range from dilution problems and theflow cytometer's inherent need to isolate and distinguish each cellindividually as well as the pressure and other stresses which typicalflow cytometry has, prior to the present invention, imposed upon thecells or other substances that it was sorting. This may also represent aunique factor for sperm cells because it appears that even though thesperm cell may appear to pass through the flow cytometer and be sortedwith no visually discernable side-effects, in fact, the cells themselvesmay have been stressed to the point that they perform less thanoptimally in the insemination process. Thus, an interplay of factorsseems involved and has raised unusual problems from the perspective ofsperm cell sorting and ultimate use for artificial insemination.

Another problem which has remained—in spite of the great advancesachieved through the Johnson patent and related technology—is the factthat prior to the present invention it has been extremely difficult toachieve lower dosage insemination with sexed sperm. While historically,some achievement of low dose insemination has occurred, it has appearedto be more on a theoretical or laboratory environment rather than fromenvironments which are likely to be experienced in or applicable to acommercial application. In this regard, the desire has not been merelyto achieve low dose insemination but rather to achieve low doseinsemination with pregnancy success rates which are comparable toexisting unsexed, high dosage artificial insemination efforts. Thus, theadvances achieved by the present inventors in both sexed and low doseartificial insemination represent significant advances which may, forthe first time, make commercial applications feasible.

Another problem which has been faced by those in the industry—again, inspite of the great advances by the Johnson patent and relatedtechnology—is the fact that the problem itself, namely, artificialinsemination with a high success rate is one of a statistical nature inwhich a multitude of factors seem to interplay. Thus, the solutionsproposed may to some degree involve a combination of factors which, whenthoroughly statistically studied, will be shown to be necessary eitherin isolation or in combination with other factors. Such a determinationis further compounded by the fact that the results themselves vary byspecies and may be difficult to ascertain due to the fact that testingand statistical sampling on a large enough data base is not likely to beworth the effort at the initial stages. For these reasons the inventioncan also involve a combination of factors which may, individually or incombination, represent the appropriate solutions for a givenapplication. This disclosure is thus to be considered broad enough sothat the various combinations and permeations of the techniquesdisclosed may be achieved. Undiscovered synergies may exist with otherfactors. Such factors may range from factors within the sorting or flowcytometer steps to those in the collection as well as inseminationsteps. At present, studies have been primarily achieved on bovinespecies, however, it is not believed that these techniques will belimited to such species or, for that matter to only sperm cells. Itappears that the techniques used may have application beyond just spermcells into areas which involve either sensitive items to be sorted ormerely minimization of the impacts of the stresses of flow cytometryupon the item sorted.

Interestingly, while the present invention takes an approach to minimizethe impacts and stresses upon the sperm cells, others appear to haveactually taken steps away from this direction by increasing pressuresand demands for speed and other such performance. Essentially, the drivefor low dose insemination and high speed processing may, in anindividual or perhaps interrelated fashion have posed problems whichlimited one another. Thus, while there has been a long felt butunsatisfied need for high speed, low dose sexed insemination, and whilethe implementing arts and elements have long been available, prior tothe present invention the advances or perhaps combinations of advanceshad apparently been overlooked by those skilled in the art. Perhaps tosome degree they failed to appreciate that the problem involved aninterplay of factors as well as peculiar necessities for the types ofcells (sperm cells or perhaps species-specific sperm cells) involved inthis field. Interestingly, as the listing of efforts earlier in thisdiscussion shows, substantial attempts had been made but they apparentlyfailed to understand the problem inherent in such an area as low dose,sexed insemination and had perhaps assumed that because the naturalservice event involves perhaps billions of sperm, there may have beenphysical limitations to the achievement of artificial insemination withnumbers which are as many as four orders of magnitude less in number.Thus, it may not be surprising that there was to some extent an actualteaching away from the technical direction in which the presentinventors went. Perhaps the results may even be considered unexpected toa degree because they have shown that sexed, low dose artificialinsemination can be achieved with success rates comparable to those ofunsexed, high dose artificial insemination. It might even be surprisingto some that the techniques and advances of the present invention infact combine to achieve the great results shown. While each techniquecould, in isolation, be viewed by some as unremarkable, in fact, thesubtle changes appear to afford significant advances in the endresult—whether considered alone or in combination with other subtlechanges.

Thus, until the present invention the achievement of success rates forlow dose, sexed artificial insemination has not been possible withlevels of performance necessary or simplified procedures likely to benecessary to achieve commercial implementation. The present inventiondiscloses techniques which permit the achievement of improvedperformances and thus facilitate the end result desired, namely, lowdose, sexed artificial insemination on a commercial basis.

SUMMARY OF THE INENTION

Accordingly, the present invention provides improved sheath andcollector systems for sorting of sperm cells to determine their sexthrough a flow cytometer. The sheath fluid as typically used in a flowcytometer is replaced with a fluid which minimizes the stress on thesperm cells as they are sorted. Furthermore, the collection system isimproved to minimize both the physical and chemical stress to which thesperm cells are subjected. Various techniques and substances arerepresented but as those skilled in the art will readily understand,various combinations and permutations can be used in the manner whichmay be optimized for performance based in the species, goals and otherparameters involved in a specific processing application.

An object of the invention is thus to achieve better sorting forsubstances such as sperm cells. A goal is to minimize the impact thesorting function itself has on the cells or other sensitive items whichmay be sorted. A particular goal is to minimize the impact the sheathfluid imposes upon the cells and to potentially provide a sheath fluidwhich affirmatively acts to assist the cells in handling the variousstresses involved. A parallel goal is to provide substances andtechniques which are especially suited for sperm cells in general, forbovine sperm cells, for equine sperm cells, and for the separation ofsuch sperm cells into X- and Y-chromosome bearing components. Similarlya goal is to minimize the impacts that the collection phase (e.g., aftersorting) has upon the cells and to minimize the physical impact as wellas chemical impacts on such sex sorted sperm cells. Thus a goal is toachieve as unaffected a sorted result as possible.

Another object of the invention is to achieve low dose, sortedinsemination on levels and with success rates which are comparable tothose of the typical unsexed, high dose artificial insemination. Thusthe prior goals of minimizing the stress or potential damage upon thesperm cells is important. Sorting in a manner which affords both highspeed and low stress sorting, and which is especially adapted for spermcell sorting in a low dose context is an important goal as well. Thegoals of providing sheath and other fluids which do not negativelyaffect the fertility of the sperm and which are compatible withartificial insemination are also important.

Naturally further objects of the invention are disclosed throughoutother areas of the specification and claims.

BRIEF DESCRIPTON OF DRAWINGS

FIG. 1 is a schematic diagram of a sorter system according to thepresent invention.

FIG. 2 is a diagram of the entrained cells in the free fall area of atypical flow cytometer.

FIG. 3 is a conceptual diagram showing differences as they roughlyappear as a result of the present invention.

FIG. 4 is a diagram of the sorted cell stream as they are collected inthe landing zone area.

DETAILED DESCRIPON OF THE PREFERRED EMBODIMENT

As will be seen, the basic concepts of the present invention can becombined and embodied in a variety of ways. The invention involves bothimproved flow cytometer systems as well as systems for the creation ofsex-specific sperm samples which may be used in artificial inseminationand the animals produced by such techniques. Furthermore, the techniquesare disclosed in a general fashion so that they may be applied tospecific systems and applications once the general principals areunderstood. While device enhancements are disclosed it should beunderstood that these enhancements not only accomplish certain methodsbut also can be varied and combined in a number of ways. Importantly, asto all of the foregoing, each of these facets should be understood to beencompassed by this disclosure.

As mentioned, the basic goal is that of separating the X-bearing spermfrom the Y-bearing sperm. This is done in a manner which isolates thetwo types of sperm so that each can be separately packaged and dealtwith. The isolation is preferably done through the use of flowcytometry. Flow cytometry in general is a technique which is wellunderstood. For instance, the basic aspects of it are shown anddiscussed in a variety of patents to Cytomation, Inc. such as the U.S.Patents and other publications listed earlier. Each of these patents andthe references cited therein, are incorporated by reference, thus thoseskilled in the art can easily understand the basic principles involved.

Essentially, flow cytometry involves sorting items, such as cells, whichare provided to the flow cytometer instrument through some type of cellsource. A conceptual instrument is shown in FIG. 1. The flow cytometerinstrument includes a cell source (1) which acts to establish or supplycells or some other type of item to be analyzed by the flow cytometer.The cells are deposited within a nozzle (2) in a manner such that thecells are surrounded by a sheath fluid (3). The sheath fluid (3) isusually supplied by some sheath fluid source (4) so that as the cellsource (1) supplies its cells, the sheath fluid (3) is concurrently fedthrough the nozzle (2). In this manner it can be easily understood howthe sheath fluid (3) forms a sheath fluid environment for the cells.

Since the various fluids are provided to the flow cytometer at somepressure, they flow out of nozzle (2) and exit at the nozzle orifice(5). By providing some type of oscillator (6) which may be veryprecisely controlled through an oscillator control (19), pressure wavesmay be established within the nozzle (2) and transmitted to the fluidsexiting the nozzle (2) at nozzle orifice (5). Since the oscillator (6)thus acts upon the sheath fluid (3), the stream (7) exiting the nozzleorifice (5) eventually and regularly forms drops (8). Because the cellsare surrounded by a sheath fluid environment, the drops (8) may containwithin them individually isolated (generally) cells or other items.

Since the drops (8) generally contain isolated cells, the flow cytometercan distinguish and separate droplets based upon whether or not theappropriate cell or cells is/are contained within the drop. This isaccomplished through a cell sensing system (9). The cell sensing systeminvolves at least some type of sensor (10) which responds to the cellscontained within each drop (8) as discussed at length in the seminalwork (no pun intended) by Larry Johnson, namely, U.S. Pat. No.5,135,759. As the Johnson patent explains for sperm cells, the cellsensing system (9) may cause an action depending upon the relativepresence or relative absence of a particular dye which may be excited bysome stimulant such as the laser exciter (11). While each type of spermcell is stained by the dye, the differing length of the X-chromosome andthe Y-chromosome causes different levels of staining, Thus, by sensingthe degree of dye present in the sperm cells it is possible todiscriminate between X-bearing sperm and Y-bearing sperm by theirdiffering emission levels.

In order to achieve the ultimate separation and isolation of theappropriate cells, the signals received by sensor (10) are fed to sometype of sorter discrimination system (12) which very rapidly makes thedecision and can differentially charge each drop (8) based upon whetherit has decided that the desired cell does or does not exist within thatdrop (8). In this manner the sorter discrimination system (12) acts topermit the electrostatic deflection plates (13) to deflect drops (8)based on whether or not they contain the appropriate cell or other item.As a result, the flow cytometer acts to sort the cells by causing themto land in one or more collectors (14). Thus by sensing some property ofthe cells or other items the flow cytometer can discriminate betweencells based on a particular characteristic and place them in theappropriate collector (14). In the system presently used to sort sperm,the X-bearing sperm droplets are charged positively and thus deflect inone direction, the Y-bearing sperm droplets are charged negatively andthus deflect the other way, and the wasted stream (that is unsortablecells) is uncharged and thus is collected in an undeflected stream intoa suction tube or the like.

Referring to FIG. 2, the process can be even further understood. Asshown in that figure, the nozzle (2) emits a stream (7) which because ofthe oscillator (6) (not shown in FIG. 2) forms drops (8). Since the cellsource (1) (not shown in FIG. 2) may supply sperm cells (15) which havebeen stained according to the Johnson technique, the light stimulationby laser exciter (11) is differentially determined by sensor (10) sothat the existence or nonexistence of a charge on each drop (8) as itseparates from stream (7) can be controlled by the flow cytometer. Thiscontrol results in positively charged, negatively charged, and unchargeddrops (8) based upon their content. As shown in FIG. 2, certain dropsare shown as deflected drops (16). These deflected drops (16) are thosecontaining sperm cells (15) of the one or the other sex. They are thendeposited in the appropriate collector (14) for later use.

One of the aspects of flow cytometry which is particularly important toits application for sperm sorting is the high speed operation of a flowcytometer. Advances have been particularly made by the flow cytometersavailable through Cytomation, Inc. under the MoFlo® trademark. Theseflow cytometers have increased sorting speeds extraordinarily and havethus made flow cytometry a technique which is likely to make feasiblethe commercial application of sperm sorting (among other commercialapplications). They act to achieve high speed sorting, that is at aspeed which is notably higher than those otherwise utilized.Specifically, Cytomation's MoFlo® flow cytometers act with oscillatorfrequencies of greater than about five kilohertz and more specificallycan be operated in the 10 to 30 or even the 50 kilohertz ranges. Thusdroplets are formed at very high frequencies and the cells containedwithin the sheath fluid environment can be emitted very rapidly from thenozzle (2). As a result, each of the components such as the nozzle (2)oscillator (6), and the like which make up and are part of a flowcytometer system result in a high speed cell sorter. In the applicationof a high speed cell sorter to the sorting of sperm cells, sorting atrates of greater than about 500 sorts per second is achieved. In fact,rates of sorting in the thousand and twelve hundred ranges have alreadybeen achieved through a high speed cell sorter. Importantly, it shouldbe understood that the term “high speed” is a relative term such that asother advances in flow cytometry and specific applications are achieved,the aspect which is considered “high” may be varied or may remainabsolute. In either definition, the general principle is that thesorting may occur at rates at which the parameters and physicalcharacteristics of the flow cytometer are significant to the cellsthemselves when sorting particular cells such as sperm cells.

One aspect of high speed sorting which appears to come into play whensorting sperm cells is that of the pressures and other stresses to whichthe sperm cells are subjected within the flow cytometer. For instance,when operating at high speeds (and an alternative definition of “highspeed”), flow cytometers can be operated at a pressure of 50 pounds persquare inch and even 60 and higher pounds per square inch. Thesepressures may be considered high because they may result in effects uponthe cells being sorted. The key as disclosed in the present inventionfor this facet is the fact that the stress thresholds of the particularcells are the determining factor. Additionally as further knowledge isgained it may be shown that the stress thresholds are a function ofcombined effects such as the particular species or the particular prioror subsequent handling of the cells. The key in this regard is that thestress imposed upon the cells can, in fact, alter their viability andtheir ability to achieve the desired result. In the pressure case, itmay be that merely subjecting the sperm cells to a higher pressure as aresult of the operation of the flow cytometer at that pressure mayresult in decreased performance of the cells. The present invention inone regard acts to minimize these stresses and thus results in greaterefficacies as well as lower dosages as discussed later.

In considering the stress aspect of the cells, the present inventionacts in a fashion which minimizes the stresses. These stresses can beminimized at any point in the over all cycle or process of collecting,sorting or even inseminating the animal. Importantly, the stress imposedby the handling of the cells within the flow cytometer appearssignificant for this application. In one embodiment of the invention,the sheath fluid is specifically selected so that it can serve in acoordinated fashion with both (or either) the pre-sort cell fluidenvironment or the post-sort cell fluid environment. While naturally itis possible to adjust either the pre- or post-sort fluids, in oneembodiment the invention adjusts the sheath fluid (3) so that it imposessignificantly less stress upon the cells than was previouslyaccomplished. In one regard the invention is remarkable in that itremoves the total focus from that of operation of the flow cytometer toa focus on handling and removing stress from the cells themselves. Forinstance, while it has been known to utilize fluids having a proper pHfactor or osmoality, the present invention recognizes that there may becertain chemical compositions to which the cells may behyper-responsive. These hyper-responsive chemical compositions maynaturally vary based upon the cells or even the prior handling of thecells. Importantly at present it appears that for sperm cells certainmetabolic chemical compositions such as citrate seem to preventunusually high stresses upon the cells. Thus, the hyper-responsivechemical compositions can be defined as those to which the cells areparticularly responsive in the context of their functionality and thethen-existing handling techniques. As to sperm cells it appears thatmetabolic compositions, specifically citrate constancy for bovine spermcells and hepes buffer constancy for equine sperm cells may be veryimportant. Thus the present invention acts to minimize the changesthrough the type of operation or the selection of substances which mayact as a means for minimizing the changes which the cells experience.

For the sheath fluid, a substance is selected according to oneembodiment of the invention so that it may be chemically coordinated toprevent minimal changes. Thus, by selecting the appropriate sheath fluidnot only in context of flow cytometry parameters, but rather also incontext of the cell parameters themselves, the changes experienced bythe cells and the over all result of the sorting can be enhanced. Thisis shown conceptually in FIG. 3. FIG. 3 shows some type of chemicalfactor (such as citrate or other factors) as it may exist throughout thevarious phases of the process. For instance, the four phases shown mightrepresent the following: phase I may represent the existence of thecells within the cell source (1), phase II might show the existence ofthe cells as they are sorted in the sheath fluid environment, phase IIImight show the cells as they are collected after sorting and phase IVmight show the reconstituted cells in a storage medium after sorting.These four phases as shown for the prior art may experience vastlydifferent chemical factor environments. As shown conceptually, however,in the present invention the cells may experience very little change,most notably the dip or drop experienced between phases I and II may bevirtually absent. This is as a result of the selection of theappropriate sheath fluid as mentioned above. Thus, as a result of beingsubjected to an appropriate sheath fluid, the cells in the presentinvention may experience a much lower level of stress.

One of the potential generalities that may exist with respect to thisphenomenon is the fact that certain chemical compositions may representmore hyper-responsive chemical compositions than others. While naturallythis may vary based upon the species of sperm, the handling, or even thetype of cell involved, it appears that the viability of the cells fortheir intended purpose (here, artificial insemination) varies greatly,naturally or because of sorting or both, and so the cells exhibit ahyper-responsive character with respect to that chemical composition. Byselecting certain metabolic chemical compositions, most notably citratesor chemicals which are within the citric acid cycle, great advancesappear possible. Thus for the bovine sperm application, the sheath fluid(3) is selected and coordinated so that it presents about a 2.9 percentsodium citrate composition. Specifically, the 2.9 percent sodium citratesolution may be created as follows:

-   -   1. Place 29.0 grams of sodium citrate dihydrate (Na₃C₆H₅O₇·2H₂O)        in a 1,000 ml volumetric flask        -   a. Dissolve sodium citrate in ¾ of water batch, then add            water to volume.    -   2. Add deionized or Nanopure water to make 1,000 ml final        volume.    -   3. Transfer to bottles and autoclave at 15 lbs pressure (245°        F.) for at least 30 minutes        -   a. Autoclave solution using conditions to minimize            evaporation (loose cover)        -   b. Be careful that water does not boil away.    -   4. Cool slowly at room temperature.    -   5. Store sealed in a 5° C. cold room.    -   Further, for a sheath fluid, the sodium citrate solution may be        filtered.    -   6. Filter with a 0.22 micron filter using aseptic techniques.

Interestingly, for equine sperm cells such a composition does notperform as well. Rather, it has been discovered that for equine spermcells, a hepes buffered medium such as a hepes bovine gametemedium—particularly HBGM3 as previously created by J. J. Parrish for abovine application—works well. This medium is discussed in the article“Capacitation of Bovine Sperm by Heparin”, 38 Biology of Reproduction1171 (1988) hereby incorporated by reference. Not only is thissurprising because it is not the same type of substance as is utilizedfor bovine sperm, but the actual buffer, originally was developed for abovine application. Thus in the equine application the sheath fluid isselected which contains the hepes buffer. This solution may have a pH atroom temperature of about 7.54 (pH at 39° C.=7.4) with the followingcomposition:

Chemical Dry weight (g/500 ml) CaCl₂ 0.145 KCl₂ 0.115 MgCl₂•6H₂O 0.004NaH₂PO₄•H₂O 0.018 NaCl 2.525 NaPyruvate 0.011 Lactic Acid (60%) 1.84 mlHEPES 4.765 NaHCO₃ 0.420 BSA (fraction V) 3.0

One other aspect which may interplay in the present invention is thefact that the cells involved may experience unusual sensitivities. Inone regard this may be due to the fact that sperm cells are in a classof cells which are non-repairing cells. That is, they do not have theability to repair themselves and hence, they may need to be treated muchmore sensitively than is typical for flow cytometers or other handlingequipment. Thus, it may be appropriate that the enhancement isparticularly applicable when the flow cytometer acts to establish asource of sperm cells. Another potentially related aspect which may beunique to a class of cells such as sperm cells is the fact that theirDNA is non-repairing, non-replicating, and non-transcribing. Either ofthese factors may come into play and so they may be relevant eitherindividually or together. Thus, it may be that the teachings of thepresent invention apply to all gamete cells or even to viruses and thelike which are non-repairing, non-translating, non-transcribing cells.

A separate aspect of the flow cytometer processing which may also beimportant is the fact of properly treating the cells both chemically andphysically after they are sorted. As shown in FIG. 4, as the cellswithin drops (8) land in collector (14), it may be important that thecontainer which makes up the collector be properly sized so that it actsas some means of avoiding an impact between the cells and the containeritself While it has been known to place an initial collector fluid (17)in the bottom of the container to collect the cells so that they do nothit the bottom of the container, it appears that a simple widening ofthe container to address variations in stream presentation as well asthe inevitable splashing due to the impact of the cells into thecontainer can be used to enhance the result. In one regard this can actas a cushioning element so that cells which may be mechanicallydelicate, that is, they may break or be damaged by an impact can betreated appropriately. Thus when the cytometer source establishes cellswhich are physically delicate cells as the cells to be sorted, it may beimportant to provide some type of cushioning element such as a widecollection tube for which the opening width (18) serves to position thewalls of the container in a manner which avoids contact with the cells.Thus the tube does not present side walls so close that there is anysignificant probability of contact between those cells being sorted andthe walls of the tube. In this manner, in addition to the collectorfluid (17), it may be desirable to include a wide collection tube aswell. Perhaps merely providing a wide opening to the container whichserves as part of the collector (14) may be sufficient. For applicationsutilizing high speed sorting of sperm cells, it has been found thatproviding a container having an inner diameter opening of at least 15millimeters is believed to be sufficient. Specifically when utilizing a14 ml Falcon test tube in such an application, minimal physical damageto the cells as a result of the collector (14) has been discovered.

It should be noted that even the 14 ml Falcon test tube may not beoptimum. Specifically, it is believed that designing a collectioncontainer which matches the geometry of the stream (that is, a“stream-matched container”) may be most optimal. This stream-matchedcontainer may have any or all of the following characteristics: arelatively wide orifice, an elliptically shaped orifice, a lesser heightto width ratio than currently involved, an angled or otherwisecoordinated presentation such as may present side walls which areparallel to the falling streams, and the like. It may also be desirableto provide a mounting element such as a movable element or medium likeball bearings or the like to permit variable orientation of the tube tomatch the falling stream desired to be collected. In addition, thephysical characteristics for the class of containers such as theexisting tube (described as a “Falcon-type” test tube) may include notonly the width of the tube but also the material (such polystyrene towhich the cells do not stick) out of which it is made and the like.(These material options are well known for the 14 ml Falcon tube.) Thusthe container and it collection fluid may also serve as a cushioningelement to minimize physical damage to the cells. It also can serve, byits size, to facilitate collection of adequate numbers of sperm withouta significant dilution effect.

Another aspect of the collector fluid (17) can be the fact that it, too,may serve to minimize chemical stresses upon the cells. In one regard,since it may be important to provide a nutrient to the cells both beforeand after sorting, the collector fluid (17) may be selected so as toprovide a coordinated level of nutrient so that the levels are balancedboth before and after sorting. For bovine sperm in which a nutrient ofegg yolk citrate is utilized at a two percent egg yolk level, it hasbeen discovered that utilizing a six percent egg yolk citrate level(that is six percent egg yolk content in a citrate solution) providesgood results. This is as result of the volumes existing before and afterthe sorting event. The collector fluid (17) may start (before sorting)with about 2 ml of volume. The sorting event may add about double thisvolume (ending at three times the initial starting volume) with verylittle egg yolk citrate in solution (due to clogging and other flowcytometer considerations). Thus, the end result in terms of the level ofthe amount of egg yolk citrate present may be equivalent to the startingresult, namely, two percent egg yolk content in a citrate solution dueto the volumes involved. Thus the collector fluid (17) may be selectedso as to create an ending collector fluid environment which is balancedwith the initial nutrient or other fluid environment. In this manner, itmay serve to minimize the time and changed level of composition to whichthe cells are subjected. Naturally, these fluid environments may bepresented within the flow cytometer or may exist at some other priortime, the important point being merely minimizing the stress to whichthe cells are subjected at any time in their life cycle. Furthermore,since the initial chemical substance content can be varied (for instancethe percent egg yolk content in the citrate may be varied up or down),likewise the starting collection fluid environment or various volumesmay also be varied so that the ending result is the same. Thus, prior tocommencing the sorting process, the collector fluid exists with a sixpercent egg yolk content in the citrate solution and after completion ofthe sorting event the collector fluid—with the sex-specific sperm—mayresult in a two percent egg yolk content in the citrate solution similarto the initial nutrient content.

Note that in later use these sperm cells may be treated to a 20% eggyolk content in the citrate fluid for other reasons, however thesechanges are not deemed to provide stress to the cells as they are merelya known part of the total insemination process. While naturally thelevels may be varied as those skilled in the art readily understand, a20% egg yolk citrate buffer may be constituted as follows:

I. FINAL COMPOSITION:

80% sodium citrate solution (72 mM)

20% (vol/vol) egg-yolk

II. PREPARATION FOR 1 LITER:

A. Sodium citrate solution

-   -   1. Place 29.0 grams of sodium citrate dihydrate (Na₃C₆O₇·2H₂O)        in a 1,000 ml volumetric flask    -   2. Add deionized or Nanopure water to make 1,000 ml final        volume.    -   3. Transfer to bottles and autoclave at 15 lbs pressure (245°        F.) for at least 30 minutes.        -   a. Autoclave solution using conditions to minimize            evaporation (loose cover)        -   b. Be careful that water does not boil away.    -   4. Cool slowly at room temperature.    -   5. Store sealed in a 5° C. cold room.

B. Egg preparation

-   -   1. Obtain fresh hen's eggs from a good commercial source.    -   2. Wash the eggs free of dirt ('do not use too much detergent)        and rinse.    -   3. Immerse eggs in 70% ethanol for 2-5 minutes.    -   4. Remove eggs and allow to dry (or wipe dry) and store on a        clean towel.

C. Preparation of extender

-   -   1. Use sterile, clean glassware    -   2. A-fraction (non-glycerol fraction)        -   a. Place 800 ml of 2.9% sodium citrate solution in a 1,000            ml graduated cylinder.        -   b. Antibiotic levels for the non-glycerol containing            fraction (A-fraction) of the extender may be as follows:            -   I. Tylosin=100 μg/ml            -   ii. Gentamicin=500 μg/ml            -   iii. Linco-spectin=300/600 μg/ml        -   c. Add 200 ml of fresh egg-yolk as outlined below (Section            D)            -   I. Mix very thoroughly.        -   d. This provides A-fraction extender based on 2.9% sodium            citrate, with 20% egg-yolk and antibiotics at concentrations            known to be non-toxic to bull sperm.        -   e. Extender can be stored overnight at 5° C.        -   f. Decant supernatant (upper 800 ml) the next day.        -   g. Warm to 37° C. prior to use the next day.

D. To add egg-yolk to a buffered solution, the following procedure workswell.

-   -   1. Wash egg and clean the eggs (see B above)    -   2. Open egg and separate yolk from albumin using a yolk        separator. Alternatively, pour yolk back and forth 2-3 times        between the two half shells. Do not rupture the membrane around        the yolk.    -   3. Place the yolk onto a sterile piece of 15 cm filter paper.    -   4. Hold the filter paper over the graduated cylinder containing        buffer and squeeze the yolk (rupturing the membrane) and allow        the yolk to run out of the golded filter paper into the        cylinder. Typically about 12-15 ml of the yolk can be obtained        from one egg.

Another aspect which may interplay in the various factors of the presentinvention is that of utilizing low dose amounts of sperm for artificialinsemination or the like. Additional background on the aspect of sexed,artificial insemination may be found in “Prospects for Sorting MammalianSperm” by Rupert P. Amman and George E. Seidel, Jr., Colorado AssociatedUniversity Press (1982) hereby incorporated by reference. As mentioned,natural insemination involves numbers of sperm on the order of billionsof sperm. Typical artificial insemination is presently conducted withmillions of sperm for bovine species and hundreds of millions of spermfor equine species. By the term “low dose” it is meant that the dosageof sperm utilized in the insemination event are less than one-half orpreferably even less than about 10% of the typical number of spermprovided in a typical artificial insemination event. Thus, the term “lowdose” is to be viewed in the context of the typical artificialinsemination dosage or also as an absolute number. For bovine spermwhere currently 1 to 10 million sperm are provided, a low dose processmay be considered an absolute number of about 500,000 sperm or perhapsas low as 300,000 sperm or lower. In fact, through utilization of thetechniques of the present invention, artificial insemination with goodpercentages of success has been shown with levels of insemination ofsperm at 100,000 and 250,000 sperm (41% and 50%, respectively pregnancyrates). As shown in the article “Uterine Horn Insemination of HeifersWith Very Low Numbers of Non-frozen and Sexed Spermatozoa” as publishedin 48 Theriogenology 1255 (1997) hereby incorporated by reference. Sincesperm cells appear to display a sensitivity to dilution, these resultsmay display particular interdependence on the utilization of low dosesperm samples with regards to various techniques of the presentinvention. The absolute numbers may be species dependent, for equinespecies, merely less than about ten, five, or even one million sperm maybe considered a low dose process.

Another aspect which may be important is the fact that the sperm sexedthrough the present invention techniques is utilized in an artificialinsemination system. Thus, when the collector (14) is used to providesperm for artificial insemination the techniques of the presentinvention may be particularly relevant. Further, it is possible that thecombination of both artificial insemination use and the use in a lowdose environment may together create synergies which makes the varioustechniques of the present invention particularly appropriate. Naturally,the sexed sperm can be utilized not just in an artificial inseminationmode, but in other techniques such as in vitro fertilization and thelike.

The process of collecting, sorting, and eventually inseminating ananimal through the use of flow cytometry involves a variety of steps. Inthe context of bovine insemination, first the semen is collected fromthe bull through the use of an artificial vagina. This occurs at ratesof approximately 1.5 billion sperm per ml. This neat semen may bechecked through the use of a spectrophotometer to assess concentrationand may be microscopically evaluated to assure that it meets appropriatemotility and viability standards. Antibiotics may then added. As aresult the initial sample may have approximately 60 to 70 percent of theprogressively motile sperm per ejaculate. For processing, a dilutionthrough of some type TALP (tyrode albumin lactate pyruvate) may be usedto get the numbers of sperm at a manageable level (for flow analysis) ofapproximately 100 million per ml. The TALP not only nurtures the spermcells, but it may make them hyper-activated for the staining step. Priorto staining, in some species such as the equine species, centrifugationmay be accomplished. Staining may be accomplished according to amulti-stained or single-stained protocol, the latter, the subject of theJohnson Patent and related technology. The staining may be accomplishedwhile also adjusting the extender to create the appropriate nutrientenvironment. In bovine applications this may involve addingapproximately 20% egg yolk content in a citrate solution immediatelyafter staining. Further, in staining the sperm cells, it has beendiscovered that by using higher amounts of stain than might to someextent be expected better results may be achieved. This highconcentration staining may involve using amounts of stain in the tens ofmicro-molar content such as discussed in the examples below where 38micro-molar content of Hoechst 33342 stain was used.

After adding the stain, an incubation period may be used such asincubating at one hour at 34° C. to hasten the dye uptake withconcentrations at about 100 million sperm cells per ml. Filtration maythen be accomplished to remove clumps of sperm cells and then dilutionor extending may or may not be accomplished to the desired sortconcentration of approximately 100 million sperm cells per ml may beaccomplished. Sorting according to the various techniques discussedearlier may then be accomplished from which sperm cells may be recoveredin the collection phase. As mentioned earlier, the collection may resultin samples with approximately 2% egg yolk citrate concentrate content(for bovine species). This sample may then be concentrated to about 3-5million sperm cells per ml through the use of centrifugation after whichthe sheath fluid and preserving fluid may be removed. A final extensionmay then be accomplished with either 20% egg yolk citrate or a CornellUniversal Extender or the like. The Cornell Universal Extender may havethe following composition for 1000 ml:

-   -   14.5 g sodium citrate dihydrate    -   2.1 g NaHCO₃    -   0.4 g KCl    -   3.0 g glucose    -   9.37 g glycine    -   0.87 g citric acid    -   For 20% egg-yolk        using 800 ml of above preparation and may include about 200 ml        of egg-yolk composition.

After this last extending, 3 to 5 million sperm per ml (for bovinespecies) may result. This sample may then be cooled to slow the sperm'smetabolism and to permit use over longer periods of time. In the equinespecies the sample may then be used in oviductal or other inseminationprocesses as those skilled in the art well understand. In bovine sperm,the sample may be diluted yet one more time to the desired dosage level.It has been discovered that dilution may create an effect upon the spermcell's viability and so it may be appropriate to avoid too large a levelof dilution by providing a smaller sample. At present, low dosages ofapproximately 300,000 sperm per 0.184 ml may be achieved. Furthermore,it may be desirable to maintain a level of seminal plasma atapproximately a five percent level, although the results of thisrequirement are, at present, mixed. The sperm cell specimen may then beplaced in a straw for use in artificial insemination and may betransported to the cows or heifers to be inseminated.

In order to achieve conveniently timed artificial insemination, heiferor cow estrus may be synchronized using known techniques such as theutilization of prostaglandin F2_(α) according to techniques well knownin the art. This latter substance may be particularly valuable in thatit has been reported to potentially achieve enhanced fertility inheifers as discussed in the article “Prostoglandin F2_(α)—A FertilityDrug in Dairy Cattle?”, 18 Tneriogenology 245 (1982) hereby incorporatedby reference. While recent results have not maintained this premise, itmay be that the present invention demonstrates its particular viabilityin situations of sexed, low dose insemination. For bovine species,artificial insemination may then be accomplished through the use ofembryo transfer equipment with placement of the sperm cells deep withinthe uterine horns. This may be accomplished not at the peak moment astypically used in artificial insemination, but rather at a somewhatlater moment such as 12 hours after that time since there is somepossibility that fertility for sexed artificial insemination may occurslightly later. The utilization of embryo transfer equipment may be usedbecause there may be high sensitivity of the uterine wall for such lowdose, sexed inseminations.

Further the techniques can be combined to achieve higher efficiencyproduction as well. Particularly, the processes now invented whichpermit high speed sorting and low dose insemination of sexed embryos isalso possible in a superovulated animal. The superovulation may beachieved by use of a superovulatory pharmaceutical or by any othertechnique. The superovulatory pharmaceutical may act directly orindirectly, such as through a sequence of reactions to achieve a greaterthan normal production of eggs. The combination with superovulation issurprising because superovulation was previously deemed to hinder such acombination. Sperm transport is compromised in superovulated cattle, so,animals were frequently artificially inseminated on multiple occasionsand/or with multiple doses of semen. Also, prior procedures for sexingsemen were relatively slow; therefore, it was of interest to determinefertilization rates after a single insemination of superovulatorypharmaceutical, such as FSH (follicle stimulating hormone)-treatedcattle with only 600,000 total sexed unfrozen sperm using these newercombination of techniques.

By example, twelve Angus crossbred heifers were superovulated usingstandard procedures: 6, 6, 4, 4, 2, 2, 2, and 2 mg FSH were injectedintramuscularly at half-day intervals beginning between days 9 and 12 ofthe estrous cycle; 25 and 12.5 mg prostaglandin F-2 alpha were injectedintramuscularly with the 6th and 7th FSH injections. Sperm from bulls ofunknown fertility were stained with Hoechst 33342 and then sorted usinga MoFlo® flow cytometer/cell sorter yielding 700-800 live sperm of eachsex/sec. Average sort purity was 89% of the desired sex. Sorted spermwere concentrated to 3.36×10⁶ sperm/ml by centrifugation at 650 g for 10min, cooled to 5° C., and stored 4 h. Then 184 ul were loaded in 0.25 mlplastic straws; half the dose was inseminated into each uterine horn 20to 24 h post-onset of estrus using automatic side-opening embryotransfer sheaths. Embryos were collected by standard non-surgicalprocedures at 7 or 16 days post-estrus. Results were similar between day7 and 16 collections and between X- and Y-sorted sperm Embryos wererecovered from 9 heifers. There were 52 embryos (mean, 4.3-5.3/donor) atnormal stages of development, 13 retarded embryos and 31 unfertilizedova. Forty-six embryos were sexed by PCR using primers for aY-chromosome-specific DNA sequence; 43 (93%) were of the intended sex.Although this study involved few animals, surprisingly, insemination ofsuperovulated heifers with only 600,000 total (live) sexed unfrozensperm gave similar results to conventional procedures. Variations on theabove may also be accomplished, including, but not limited to, sortingthrough other than flow cytometric means, achieving superovulation inother manners, increasing fertility in other manners, and the like.

Further, the congruence of methods of sexing sperm based on DNA content,high speed flow cytometer/cell sorters, and procedures for inseminatingheifers with fewer than 500,000 total sperm without compromisingfertility has resulted in the possibility of a viable sexed semenindustry in cattle within a few years. There will be a myriad ofapplications for sperm sexed at <85% accuracy. Perhaps the most obviousis inseminating one subset of cattle (both dairy and beef) for femaleherd replacements, and having the converse subset (both dairy and beef)bred to entirely different types of bulls to produced males for meat. Avery important subset of the above is inseminating heifers withX-chromosome-bearing sperm to produce female calves, which have a lowerincidence of dystocia than male calves, primarily due to smaller size.Furthermore, proving young dairy sires would be much more efficient witha preponderance of heifer calves. Having more than 85% heifer calvesalso makes it feasible to manage dairy cows so they average fewer thantwo surviving calves per lifetime, which is attractive because ofreducing problems associated with gestation and parturition. Single sexsystems of beef production also would become feasible, in which eachfemale replaces herself and is slaughtered between 2 and 3 years of age,thus using a much higher percentage of nutrients in the system forgrowth, and a lower percentage for maintenance. Sexed semen would beuseful for in vitro fertilization and to inseminate cows superovulatedfor embryo transfer. Frequently one sex of calves is considerably morevaluable than the other, and although accurate methods of sexing embryosare available, they are time-consuming, and half of the embryos producedare of the less valuable sex, it is surmised that accurately sexed semenwould be widely adopted for artificial insemination of cattle if thesexing surcharge were low and fertility was only minimally compromised.The percentage of beef cattle inseminated artificially likely wouldincrease substantially with sexed semen.

Interestingly, rather than inseminating within the uterine body wheresuch insemination are usually placed, by insemination deep within theuterine horn, better results may be achieved. Perhaps it is alsosurprising that the samples thus far studied have shown no differencebetween ipsi- and contra-lateral inseminations when accomplished deepwithin the uterine horn. By deep, it should be understood that theinsertion is placed well into the uterine horn using the embryo transferequipment. The fact that results do not appear significantly differentusing ipsi- and contra-lateral inseminations has led the presentinventors to propose the use of insemination in both so that the processof identifying the appropriate uterine horn may no longer be needed.

As a result of the insemination, it is of course desired that an animalof the desired sex be produce This animal may be produced according tothe systems discussed earlier through the use of the sexed spermspecimen. It should also be understood that the techniques of thepresent invention may find application in other techniques such aslaproscopic insemination, oviductal insemination, or the like.

As examples, the following experiments have been conducted. While notall use every aspect of the inventions described here, they do show theperformance enhancements possible through differing aspects of theinvention Further, a summary of some experiments is contained in thearticle “Uterine Horn Insemination of Heifers With Very Low Numbers ofNon-frozen and Sexed Spermatozoa” as referenced earlier. This articlesummarizes some of the data showing the efficacy of the presentinvention. As to the experiments, one has been conducted with sexed,unfrozen sperm cells with high success as follows:

EXAMPLE 1

Angus heifers 13-14 mo of age and in moderate body condition, weresynchronized with 25 mg of prostaglandin F-2 alpha at 12-day intervalsand inseminated 6-26 h after observed standing estrus. Freshly collectedsemen from three 14-26 mo old bulls was incubated in 38 μM Hoechst 33342at 75×10⁶ sperm/ml in a TALP medium for 1 h at 34° C. Sperm were sortedby sex chromosomes on the basis of epiflourescence from laser excitationat 351 and 364 nm at 150 mW using a MoFlo® flow cytometer/cell sorteroperating at 50 psi and using 2.9% Na citrate as sheath fluid. Xchromosome-bearing sperm (˜90% purity as verified by resorting sonicatedsperm aliquots) were collected at ˜500 live sperm/sec into 2-mlEppendorf tubes containing 100 μl Cornell Universal Extender (CUE) with20% egg yolk. Collected sperm were centrifuged at 600×g for 10 min andresuspended to 1.63×10⁶ live sperm/ml in CUE. For a liquid semen unsexedcontrol; Hoechst 33342-stained sperm were diluted with sheath fluid to9×10⁵ sperm/ml and centrifuged and resuspended to 1.63×10⁶ progressivelymotile sperm/ml in CUE. Sexed semen and liquid control semen were cooledto 5° C. over 75 min and loaded into 0.25-ml straws (184 ul/straw).Straws were transported at 3 to 5° C. in a temperature-controlledbeverage cooler 240 km for insemination 5 to 9 h after sorting. Sexedsemen and liquid control semen were inseminated using side-opening bluesheaths (IMV), one half of each straw into each uterine horn (3×10⁵ livesperm/heifer). As a standard control, semen from the same bulls had beenfrozen in 0.5-cc straws by standard procedures (mean 15.6×10⁶ motilesperm/dose post-thaw), thawed at 35° C. for 30 sec, and inseminated intothe uterine body. Treatments were balanced over the 3 bulls and 2inseminators in a ratio of 3:2:2 inseminations for the sexed semen andtwo controls. Pregnancy was determined ultrasonically 31-34 days afterinsemination and confirmed 64-67 days later when fetuses also were sexed(blindly). Data are presented in the table.

No. Heifers No. Pregnant No. Pregnant No female Treatment bred d31-34d64-67 fetuses Sexed semen 45 20 (44%)  19 (42%) 18 (95%)^(a) Liquidcontrol 28 15 (54%) 15 (54%)  8 (53%)^(b) Frozen control 29 16 (55%)  15(52%) 12 (80%)^(c) ^(a,b)Sex ratios of values with differentsuperscripts differ (P <0.02).Although the pregnancy rate with sexed semen was only 80% of controls,this difference was not statistically significant (>0.1). One pregnancywas lost by 64-67d in each of the sexed and dozen control groups; 18 of19 fetuses (95%) were female in the sexed group, and 20 of 30 (67%) werefemale in the control groups. The liquid semen control yielded avirtually identical pregnancy rate to the frozen semen controlcontaining over 50 times more motile sperm (over 120 times more totalsperm), demonstrating the efficacy of low-dose insemination into theuterine horns. We have altered the sex ratio in cattle significantlyusing flow cytometer technology and artificial insemination.Similarly, an experiment was conducted with unsexed, unfrozen spermcells and may be reported as follows:

EXAMPLE 2

The objective was to determine pregnancy rates when heifers areinseminated with extremely low numbers of frozen sperm under ideal fieldconditions. Semen from three Holstein bulls of above average fertilitywas extended in homogenized milk, 7% glycerol (CSS) extender plus 5%homologous seminal plasma to 2×10⁵, 5×10⁵ or 10×10 ⁶ (control) totalsperm per 0.25 ml French straw and frozen in moving liquid nitrogenvapor. Semen was thawed in 37° C. water for 20 sec. Holstein heifers13-15 mo of age weighing 350-450 kg were injected with 25 mgprostaglandin F-2-alpha (Lutalyse®) twice at a 12-day interval andinseminated with an embryo transfer straw gun and sideopenig sheath,half of the semen deep into each uterine horn 12 or 24 h after detectionof estrus. The experiment was done in five replicates over 5 months, andbalanced over two insemination technicians. Ambient temperature atbreeding was frequently −10 to −20° C., so care was taken to keepinsemination equipment warm. Pregnancy was determined by detection of aviable fetus using ultrasound 40-44 days post-estrus and confirmed 55-62days post-estrus; 4 of 202 conceptuses were lost between these times.Day 55-62 pregnancy rates were 55/103 (53%), 71/101, (70%), and 72/102(71%) for 2×10⁵, 5 ×10⁵ and 10×10⁵ total sperm/inseminate (P<0.1).Pregnancy rates were different (P<0.05) among bulls (59, 62, and 74%),but not between technicians (64 and 65%) or inseminations timespost-estrus (65% for 12 h and 64% for 24 h, N=153 at each time). Withthe methods described, pregnancy rates in heifers were similar with5×10⁵ and 10×10⁶ total sperm per inseminate.

Prior a experiment has also been conducted on sexed, unfrozen spermcells and may be reported as follows:

EXAMPLE 3

Semen was collected from bulls at Atlantic Breeders Cooperative, diluted1:4 with a HEPES-buffered extender ÷0.1% BSA, and transported 160 km (˜2HR) to Beltsville, Md. where it was sorted at ambient temperature byflow cytometry into a TEST yield (20%) extender using methods describedpreviously (Biol Reprod 41:199). Sorting rates of up to 2×10⁶ sperm ofeach sex per 5-6 h at ˜90% purity were achieved. Sperm were concentratedby centrifugation (300 g for 4 min) to 2×10⁶ sperm/ml. Some sperm weresorted into extender containing homologous seminal plasma (finalconcentration, 5%). Sorted sperm were shipped by air to Colorado (˜2,600km) and stored at either ambient temperature or 5° C. (cooled duringshipping over 6 hr in an Equitainer, an insulated device with anice-containing compartment). Heifers or dry cows detected in estrus 11to 36 h earlier were inseminated within 9 to 29 h of the end of thesperm sorting session. Sperm (1 to 2×10⁵ in 0.1 ml) were deposited deepin the uterine horn ipsilateral to the ovary with the largest follicleas determined by ultrasound at the time of insemination

None of 10 females became pregnant when inseminated with sperm shippedand stored at ambient temperature. Of 29 females inseminated with spermcooled to 5° C. during shipping, 14 were pregnant at 4 weeks ofgestation, and 12 (41%) at 8 weeks. Eleven of the 22 inseminated within10 h of the end of sorting were pregnant at 8 weeks, but only 1 of 7inseminated 17-24 h after sorting was pregnant. There was no significanteffect of adding seminal plasma. One of the 12 fetuses was not of thepredicted sex, one was unclear, and 10 were of the predicted sex, asdetermined by ultrasonography at 60-70 days of gestation.

Subsequently, 33 additional heifers were inseminated with 0.05 ml (semenextended as described above) into each uterine horn without usingultrasonography, only 3 were pregnant 4 weeks after insemination, andonly 1 remained pregnant at 8 weeks. However, different bulls were usedfrom the previous group, and all inseminations were done 18-29 hpost-sorting. An additional 38 heifers were inseminated similarly (˜22 hpost-sorting) 200 km from our laboratory with sorted sperm from anotherbull; none of these was pregnant 8 weeks after insemination.

To summarize it is possible to achieve pregnancies in cattle viaartificial insemination of sperm sorted for sex chromosomes by flowcytometry, and the sex ratio of fetuses approximates that predicted byreanalysis of sorted sperm for DNA content (90%). However, pregnancyrates varied greatly in these preliminary experiments which requiredshipping sperm long distances. Fertility decreased drastically by 17 hpost-sorting but there was some confounding because different bulls wereused at the different times. Further studies are needed to determinewhether variation observed in pregnancy rates was due to bulldifferences, insemination techniques, interval between sorting andinsemination, or other factors.

Finally, an experiment also has been conducted with unsexed, unfrozensperm cells and may be reported as follows:

EXAMPLE 4

The objective was to determine pregnancy rates when heifers wereinseminated with very low numbers of sperm under ideal experimentalconditions. Semen from three Holstein bulls was extended in CornellUniversal Extender plus 5% homologous seminal plasma to 1×10⁵ or 2.5×10⁵sperm per 0.1 ml 2.5×10⁶ total sperm per 0.25 ml was used as a control.Fully extended semen was packaged in modified 0.25 ml plastic Frenchstraws to deliver the 0.1 or 0.25 ml inseminate doses. Semen was cooledto 5° C. and used 26-57 h after collection. Holstein heifers 13-15 mo ofage weighing 350-450 kg were injected with 25 mg prostaglandin F-2 alpha(Lutalyse®) at 12-day intervals and inseminated with an embryo transferstraw gun and side-opening sheath into one uterine horn 24 h alterdetection of estrus. Insemination was ipsilateral to the side with thelargest follicle determined by ultrasound 12 h after estrus; side ofovulation was verified by detection of a corpus luteum by ultrasound 7-9days post-estrus. Pregnancy was determined by detection of a fetus byultrasound 42-45 days post estrus. The experiment was done in fourreplicates and balanced over three insemination technicians. Side ofovulation was determined correctly in 205 of 225 heifers (91%);surprisingly, pregnancy rates were nearly identical for ipsilateral andcontralateral inseminates. Pregnancy rates were 38/93 (41%), 45/87(52%), and 25/45 (56%) for 1×10⁵, 2.5×10⁵ and 2.5×10⁶ sperm/inseminate(P>0.1). There was a significant difference in pregnancy rate (P<0.05)among technician, but not among, bulls. With the methods described, itmay be possible to reduce sperm numbers per inseminate sufficiently thatsperm sorted by sex with a flow cytometer would have commercialapplication.

As mentioned and as can be seen from the various experiments, the fieldis statistically based and thus a variety of additional experiments maybe conducted to show the appropriate combination and limitationstrategies. Thus synergies among various affects will further beidentified, such as instances in which the dye effects and combined dyeeffects the laser excitation may be studied.

The discussion included in this application is intended to serve as abasic description. The reader should be aware that the specificdiscussion may not explicitly describe all embodiments possible; manyalternatives are implicit. It also may not fully explain the genericnature of the invention and may not explicitly show how each feature orelement can actually be representative of a broader function or of agreat variety of alternative or equivalent elements Again, these areimplicitly included in this disclosure. Where the invention is describedin device-oriented terminology, each element of the device implicitlyperforms a function. Apparatus claims may not only be included for thedevice described, but also method or process claims may be included toaddress the functions the invention and each element performs. Neitherthe description nor the terminology is intended to limit the scope ofthe claims which may be submitted. It should be understood that avariety of changes may be made without departing from the essence of theinvention. Such chances are also implicitly included in the description.They still fall within the scope of this invention. A broad disclosureencompassing both the explicit embodiment(s) shown, the great variety ofimplicit alternative embodiments, and the broad methods or processes andthe like are encompassed by this disclosure.

In addition, each of the various elements of the invention and claimsmay also be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of anembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered to be encompassed in the description of each element oraction. Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. As but one example of this aspect, the disclosure of a“collector” should be understood to encompass disclosure of the act of“collecting”—whether explicitly discussed or not—and, conversely, werethere only disclosure of the act of collecting, such a disclosure shouldbe understood to encompass disclosure of a “collector.” Such changes andalternative terms are to be understood to be explicitly included in thedescription.

Any references mentioned in the application for this patent as well asall references listed in any information disclosure filed with theapplication are hereby incorporated by reference; however, to the extentstatements might be considered inconsistent with the patenting ofthis/these invention(s) such statements are expressly not to beconsidered as made by the applicant(s).

1. A method of producing multiple embryos from a female bovine mammalcomprising: a. creating superovulation in said female bovine mammal tocreate at least two eggs in each of said female bovine mammal comprisingthe step of using an ovulatory pharmaceutical to cause multiple eggs tobe produced in said female bovine mammal further comprising the stepsof: i) injecting a dosage of follicle stimulating hormone in each ofsaid female bovine mammal a plurality of times a day; ii) administeringsaid dosage of follicle stimulating hormone with prostaglandinF-2-alpha; and wherein said step of injecting said dosage of folliclestimulating hormone in each of said female bovine mammal a plurality oftimes a day comprises injecting said follicle stimulating hormone inapproximately half day increments at a dosage level of 6, 6, 4, 4, 2, 2,2, and 2 mg between days 9 and 12 inclusive of the estrus cycle andwherein administering said dosage of follicle stimulating hormone withprostaglandin F-2-alpha comprises supplementing 25 and 12.5 mg ofprostaglandin F-2-alpha on the sixth and seventh dosages, respectively,of said follicle stimulating hormone; b. collecting sperm cells from atleast one male bovine mammal; c. staining said collected sperm cellswith Hoechst 33342; d. sorting the stained sperm cells with a flowcytometer to yield live sperm cells of a desired sex; e. concentratingsaid live sperm cells of a desired sex; f. establishing an inseminationsample having in the range of 1×10⁵ to 6×10⁵ live sperm cells of adesired sex; g. inserting said insemination sample into said femalebovine mammal, half the dose into each uterine horn of said femalebovine mammal, 20 to 24 hours post-onset of estrus for said femalebovine mammal; h. fertilizing a plurality of said eggs in said femalebovine mammal; and i. producing at least two embryos of a desired sexfrom said female bovine mammal in which a plurality of said eggs werefertilized.
 2. A method of producing multiple embryos from a femalebovine mammal as described in claim 1 and further comprising the step ofseparating sperm cells based on the amount of nuclear DNA each saidsperm cell contains.
 3. A method of producing multiple embryos from afemale bovine mammal as described in claim 1, further comprising thestep of allowing at least one said embryo to develop into an animal of adesired sex.
 4. A method of producing multiple embryos from a femalebovine mammal as described in claim 1, further comprising chemicallycoordinating a sheath fluid to create a sheath fluid environment forsaid cells which is coordinated with both a pre-sort and a post-sortcell fluid environment.
 5. A method of producing multiple embryos from afemale bovine mammal as described in claim 4, wherein chemicallycoordinating a sheath fluid to create a sheath fluid environment forsaid cells which is coordinated with both a pre-sort and a post-sortcell fluid environment comprises establishing a sheath fluid whichcontains citrate.
 6. A method of producing multiple embryos from afemale bovine mammal as described in claim 1, further comprisingcollecting said sperm cells of a desired sex and cushioning said spermcells of a desired sex from impact with a collection container which hasa wide opening.
 7. A method of producing multiple embryos from a femalebovine mammal as described in claim 1, wherein said sorting said stainedsperm cells with a flow cytometer is performed at a rate of about 500sorts per second.
 8. A method of producing multiple embryos from afemale bovine mammal as described in claim 1, wherein said sorting saidstained sperm cells with a flow cytometer yields 700-800 live sperm ofthe desired sex per second.
 9. A method of producing multiple embryosfrom a female bovine mammal as described in claim 1, wherein saidconcentrating said live sperm cells of a desired sex comprisesconcentrating the live sperm cells to a concentration of about 3×10⁶ to5×10⁶ sperm per milliliter.